苏云金芽胞杆菌母细胞裂解和晶体细胞分化的机理研究

Application of Bacillus thuringiensis (Bt) instead of chemical pesticides is very important for agriculture environmental and food safety. However, inactivation of insecticidal Bt crystal under UV light decreases its application in the field. Increasing the UV-resistance and production of crystal provides an important solution. Our previous studies found that peptidoglycan hydrolases, CwlB and CwlX were involved in mother cell lysis in the standard Bt which produce crystal in the mother cell. The hydrolase CwlX was essential for mother cell lysis. We demonstrated that a novel B. thuirngiensis LM1212 produced the crystal in non-sporulating cells (crystal cells), in which the proportion of sporulating cells and crystal cells is equal. In this study, we will compare the difference between sporulating and crystal cells in the transcription and translation level to discover the different genes in the process of cell development and differentiation by using of transcriptome and proteome methods. We will construct the regulation network for differentiation of crystal cells. We will decipher transcriptional factors and regulation network in the late sporulation of mother cells by means of transcriptome analysis. We try to figure out the regulation mechanism of cwlB and cwlX genes and the activated mechanism of mother cell lysis. The secreted mechanism of CwlB and CwlX will be studied by utilization of protein-protein interaction analysis. Our findings not only provide the new insight into development and differentiation of bacteria, but also the scientific basis for solving the problem in Bt application.

苏云金芽胞杆菌（Bt）替代化学农药的应用，对农业环境和食品安全具有重要意义，但是因杀虫蛋白晶体在紫外辐射下易失活，影响了其田间应用。提高晶体的抗紫外能力和产量是重要的突破方向。前期发现肽聚糖水解酶CwlB和CwlX参与传统Bt的母细胞（母细胞产生芽胞）裂解过程，CwlX是母细胞裂解关键的水解酶；Bt菌株LM1212在非芽胞细胞（晶体细胞）产生晶体，产胞细胞和晶体细胞分化并等量形成。本项目拟通过转录组学和蛋白质组学方法，比较LM1212产胞细胞和晶体细胞的转录和转译异同，揭示细胞发育分化的差异性因子；明确晶体细胞分化的调控网络；转录组分析明确母细胞发育晚期表达的转录因子和调控网络；确定CwlB和CwlX的转录调控机制，从而解析母细胞裂解的启动机制；通过蛋白质-蛋白质互作分析明确CwlB和CwlX的分泌机制；预期结果不仅可以揭示细菌发育分化的生物学理论，同时为有效解决Bt应用问题提供科学依据。

苏云金芽胞杆菌（Bt）替代化学农药的应用，对农业环境和食品安全具有重要意义，但是因杀虫蛋白晶体在紫外辐射下易失活，影响了其田间应用。提高晶体的抗紫外能力和产量是重要的突破方向。前期发现肽聚糖水解酶CwlB和CwlC参与传统Bt的母细胞（母细胞产生芽胞）裂解过程，CwlC是母细胞裂解关键的水解酶；Bt菌株LM1212在非芽胞细胞（晶体细胞）产生晶体，产胞细胞和晶体细胞分化并等量形成。.本项目在此基础上，在两个方向开展研究，其一，通过转录组学等方法，比较分析了SigmaK和GerE突变体转录情况，解析了母细胞晚期的调控网络。阐明了CwlC具有水解酶的功能，cwlC基因的缺失使母细胞不再发生裂解，但不影响芽胞和晶体的形成、不影响对小菜蛾的杀虫活性。通过pull-down实验“钓取”到CwlC的互作蛋白PBP（青霉素结合蛋白），PBP对母细胞裂解有抑制作用。发现一个新的未知功能基因mclX，其缺失突变体的母细胞不裂解。MclX蛋白不能水解细胞壁，但是会明显抑制CwlC水解细胞壁的速率。以上结果，为母细胞裂解机制的阐明打下了坚实的基础；其二，通过LM1212转录组数据分析，确定了细胞分化的起始时间，明确了不对称隔膜形成阶段和芽胞形成起始阶段差异表达的基因。通过质粒基因突变体筛选和基因组测序，筛选到一个大多数细胞是芽胞形成细胞的LM1212突变株和一个大多数细胞是晶体产生细胞的LM1212突变株，为Bt的遗传改良提供了材料；发现了一种新的转录因子CpcR调控细胞分化和在非芽胞细胞调控cry基因表达， CpcR可显著地降低芽胞形成细胞的数量，在HD73菌株中异源验证了晶体细胞分化系统，CpcR可在非芽胞细胞中指导杀虫晶体蛋白的表达。这些结果不仅揭示了细菌发育分化的生物学理论，同时为有效解决Bt应用问题提供科学依据。